A. Gómez et col.
256
(mtROSp), mitochondrial oxygen consumption in states 4 (resting) and 3
(phosphorylating), the amounts of the respiratory complex I, II, III and IV proteins,
the apoptosis-‐inducing factor (AIF) which is also required for the
assembly/maintenance of complex I (24), the marker of oxidative damage to
mitochondrial DNA (mtDNA) 8-‐oxo-‐7,8-‐dihydro-‐2´-‐deoxyguanosine (8-‐oxodG), five
different specific markers of protein oxidative modification: the specific protein
carbonyls glutamic and aminoadipic semialdehydes (GSA and AASA) indicating
purely protein oxidative modification, the protein glycoxidation markers
carboxyethyl lysine (CEL) and carboxymethyl lysine (CML), and the protein
lipoxidation markers CML and malondialdehyde lysine (MDAL). We also measured
the full fatty acid composition of heart and SKM mitochondrial membranes which
allowed us to calculate their global unsaturation indexes (the DBI, and the
peroxidizability index, PI). The mitochondrial biogenesis indicators peroxisome
proliferator-‐activated receptor-‐γ coactivator (PGC)-‐1α (PGC1), the mitochondrial
transcription factor (TFAM), antioxidant regulation transcription factor Nrf2
(Nuclear factor erythroid 2-‐related factor 2), and the p-‐ERK and SIRT1 signalling
proteins related to longevity (25), as well as the basal metabolic rate of the whole
animal, the rectal temperature, and the heart rate, the systolic and diastolic and mean
arterial pressures were also measured.
2. MATERIALS AND METHODS
2.1. Animals and study design
128 C57BL/6 male mice were maintained in a separate room of Complutense
University animal house at the Faculty of Medicine under SPF conditions during their
whole life or until some of them were sacrificed to measure physiological or
biochemical relevant parameters. 86 of these animals (43 Old control and 43 Old-‐AT-‐
treated) were used only to obtain the survival curves. These animals were
maintained under optimum conditions (12:12 (light-‐dark) cycle, 22°C ± 2°C and
50% ± 10% relative humidity), were left intact during their whole lifespan (except
for the AT-‐treatment) and their day of spontaneous death was recorded. The
treatment with atenolol was started at 2 months of age. A separate group of 42
animals (21 controls and 21 AT-‐treated) was established at the beginning of the
experiment in order to measure the different physiological and biochemical
parameters when reaching old age (after 16 months of experimentation). Among the
21 reporter animals of each of these two groups (control and AT-‐treated), 7 animals
were used for the ROS and oxygen consumption measurements in isolated
mitochondria, 7 animals were used for the measurement of 8-‐oxodG, and 7 animals
were used to assay the rest of the biochemical parameters after being sacrificed by
cervical dislocation. At the time of sacrifice these reporter animals had 18 months of
age. The animals in the atenolol group had free access to a solution of 0.1 g/L of
